The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating a broad spectrum of industrial processes from the horticultural to medical and allied health industries. The horticultural and related agricultural industries are particularly benefiting from the advances in recombinant DNA technology.
The floriculture industry in particular strives to develop new and different varieties of flowering plants, with improved characteristics ranging from disease and pathogen resistance to altered flower colour. Although classical breeding techniques have been used with some success, this approach has been limited by the constraints of a particular species' gene pool. It is rare, for example, for a single species to have a full spectrum of coloured varieties. Accordingly, substantial effort has been directed towards the use of recombinant DNA technology to generate transgenic plants exhibiting the desired characteristics.
The development of varieties of the major cutflower species such as carnation plants, for example, having flowers exhibiting a range of colours covering lilac, violet, purple and blue or various shades thereof, would offer a significant opportunity in both the cutflower and ornamental markets.
Flower colour is predominantly due to two types of pigment: flavonoids and carotenoids. Flavonoids contribute to a range of colours from yellow to red to blue. Carotenoids impart an orange or yellow tinge and are commonly the only pigment in yellow or orange flowers. The flavonoid molecules which make the major contribution to flow colour are the anthocyanins which are glycosylated derivatives or cyanidin, delphinidin, petunidin, peonidin, malvidin and pelargonidin, and are localised in the vacuole. The different anthocyanins can produce marked differences in colour. Flower colour is also influenced by co-pigmentation with colourless flavonoids, metal complexation, glycosylation, acylation, methylation and vacuolar pH (Forkmann, 1991).
The biosynthetic pathway for the flavonoid pigments (hereinafter referred to as the "flavonoid pathway") is well established (Ebel and Hahlbrock, 1988; Hahlbrock and Grisebach, 1979; Wiering and de Vlaming, 1984; Schram et al., 1984; Stafford, 1990). The first committed step in the pathway involves the condensation of three molecules of malonyl-CoA with one molecule of p-coumaroyl-CoA. This reaction is catalysed by the enzyme chalcone synthase (CHS). The product of this reaction, 2',4,4',6'-tetrahydroxychalcone, is normally rapidly isomerized to produce naringenin by the enzyme chalcone-flavanone isomerase (CHI). Naringenin is subsequently hydroxylated at the 3-position of the central ring by flavanone 3-hydroxylase (F3H) to produce dihydrokaempferol (DHK).
The B-ring of dihydrokaempferol (DHK) can be hydroxylated at either the 3', or both the 3' and 5' positions, to produce dihydroquercetin (DHQ) and dihydromyricetin (DHM), respectively (see FIG. 1). DHQ is an intermediate required for the production of cyanidin-based anthocyanins and DHM is an intermediate required for the production of delphinidin-based anthocyanins in the flavonoid pathway. Two key enzymes involved in this pathway are flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H). The F3'H acts on DHK to produce DHQ. The F3'5'H is a broad spectrum enzyme catalyzing hydroxylation of DHK in the 3' and 5' positions and of DHQ in the 5' position (Stotz and Forkmann, 1982), in both instances producing DHM. The pattern of hydroxylation of the B-ring of anthocyanins plays a key role in determining petal colour.
Another key enzyme is dihydroflavonol-4-reductase (DFR) which has variable substrate specificity depending on its plant source and has the potential to act on any one or more of DHK, DHQ and DHM.
Many of the major cutflower species lack the F3'5'H and consequently cannot display the range of colours, resultant from synthesis of delphinidins and derivatives thereof, that would otherwise be possible. This is particularly the case for carnations which constitute a major proportion of the world-wide cutflower market. There is a need, therefore, to modify carnation plants to generate transgenic plants which are capable of producing the F3'5'H, thereby providing a means of converting DHK and DHQ to DHM, thereby influencing the hydroxylation pattern of the anthocyanins and allowing the production of anthocyanins derived from delphinidin. Flower colour is modified as a result and a single species is able to express a broader spectrum of flower colours.